The C2 domain of the ubiquitin ligase Rsp5 is required for ubiquitination of the endocytic protein Rvs167 upon change of nitrogen source
The C2 domain of the ubiquitin ligase Rsp5 is required for ubiquitination of the endocytic protein Rvs167 upon change of nitrogen source
复制标题
氮源变化时，内吞蛋白 Rvs167 泛素化需要泛素连接酶 Rsp5 的 C2 结构域
DOI：
10.1093/femsyr/foaa058
复制
发表时间：
2020
影响因子：
3.2
通讯作者：
Takagi Hiroshi
中科院分区：
文献类型：
--
作者：
Tanahashi Ryoya;Afiah Tira Siti Nur;Nishimura Akira;Watanabe Daisuke;Takagi Hiroshi
泛素化是质膜上蛋白质内吞的关键信号。酿酒酵母泛素连接酶Rsp 5含有一个氨基端膜结合C2结构域、三个底物识别结构域和一个羧基端催化同源于E6-AP羧基端（HECT）结构域，能泛素化质膜蛋白，指导其进行内吞作用。在这里，我们研究了C2结构域在胞吞作用中下调一般氨基酸通透酶Gap 1的作用，Gap 1是酿酒酵母中氮调节的通透酶之一。首先，我们构建了几个rsp 5突变体，产生不含C2结构域或具有膜结合赖氨酸残基的氨基酸变化的Rsp 5变体。这些突变体表现出缺陷的Gap 1的内吞反应的一个优选的氮源。有趣的是，我们发现，在这些突变细胞中的Gap 1的泛素化是高度相似的野生型细胞在胞吞作用。这些结果表明，C2结构域对于内吞作用至关重要，但对于Gap 1等底物的遍在化则不然。此外，遗传和生化分析表明，内吞蛋白Rvs 167是通过Rsp 5和C2结构域的泛素化所需的有效的泛素化反应的首选氮源。在这里，我们提出了C2结构域介导的质膜透性酶的内吞作用的机制。
Ubiquitination is a key signal for endocytosis of proteins on the plasma membrane. The ubiquitin ligase Rsp5 ofSaccharomyces cerevisiae, which contains an amino-terminal membrane-binding C2 domain, three substrate-recognizing tryptophan-tryptophan (WW) domains and a carboxyl-terminal catalytic homologous to the E6-AP carboxyl terminus (HECT) domain, can ubiquitinate plasma membrane proteins directing them for endocytosis. Here, we examined the roles of the C2 domain in endocytosis for the downregulation of the general amino acid permease Gap1, which is one of nitrogen-regulated permeases inS. cerevisiae. First, we constructed severalrsp5mutants producing Rsp5 variants without the C2 domain or with amino acid changes of membrane-binding lysine residues. These mutants showed defects in endocytosis of Gap1 in response to a preferred nitrogen source. Intriguingly, we found that ubiquitination of Gap1 in these mutant cells was highly similar to that in wild-type cells during endocytosis. These results indicate that the C2 domain is essential for endocytosis but not for ubiquitination of substrates such as Gap1. Moreover, genetic and biochemical analyses showed that the endocytic protein Rvs167 was ubiquitinated via Rsp5 and the C2 domain was required for efficient ubiquitination in response to a preferred nitrogen source. Here, we propose a mechanism for the C2 domain-mediated endocytosis of plasma membrane permeases.